Clinical verification of the relationship between serum lipid metabolism and immune activity in breast cancer patients treated with neoadjuvant chemotherapy

Background Lipid metabolism has been recently reported to affect the prognosis and tumor immune activity in cancer patients. However, the effect of lipid metabolism on chemosensitivity in patients with breast cancer treated with neoadjuvant chemotherapy (NAC) remains unclear. Methods We examined 327 patients with breast cancer who were treated with NAC followed by curative surgery. The correlations between the serum levels of total cholesterol (TC) and triglyceride (TG) and the clinicopathological features, including the efficacy of NAC, neutrophil-to-lymphocyte ratio (NLR), and absolute lymphocyte count (ALC), were evaluated retrospectively. Results Serum TG levels were increased after NAC in all the subtypes, and the rate of change was the highest, especially in triple-negative breast cancer (TNBC) (21.0% → 48.1%). In addition, only TNBC patients with an objective response (OR) had significantly higher TG levels after NAC than those without (P = 0.049). Patients with a high ALC before NAC had significantly higher TG levels after NAC than patients with all breast cancer (P = 0.001), HER2-enriched breast cancer (P = 0.021), and TNBC (P = 0.008). Patients with a low NLR before NAC had significantly higher TG levels after NAC only among patients with TNBC (P = 0.025). In patients with human epidermal growth factor receptor 2-enriched breast cancer, the group with normal TC levels before NAC had significantly better OS than those with high TC levels (P = 0.013, log-rank test), and in patients with TNBC, the group with high TC levels after NAC had significantly better OS than those with normal TC levels (P = 0.014, log-rank test). Conclusions Good systemic immune activity and chemosensitivity may be associated with lipid metabolism regulated by NAC in TNBC patients. Supplementary Information The online version contains supplementary material available at 10.1186/s40001-022-00964-w.


Background
Neoadjuvant chemotherapy (NAC) is the gold standard treatment for breast cancer and its use has increased the rate of breast-conserving surgery [1,2]. In addition, the pathological complete response (pCR) after NAC is a predictor of good outcome, and its prognostic value is greatest in aggressive subtypes, human epidermal growth Page 2 of 12 Goto et al. European Journal of Medical Research (2023) 28:2 factor receptor 2 (HER2)-enriched, and triple-negative breast cancer (TNBC) [3]. These intrinsic breast cancer subtypes have a high malignancy and immune activity. We have reported previously that immune-related biomarkers, including the neutrophil-lymphocyte ratio (NLR) or tumor-infiltrating lymphocytes in biopsy specimens before NAC, are associated significantly with high pCR rates in these breast cancer subtypes [4]. Immunometabolism has become a relatively new field in cancer immunotherapy, and it has been recognized that the regulation of metabolism can enhance antitumor immunity [5][6][7]. In the case of lipid metabolism, obesity has been shown to be associated positively with breast cancer risk in postmenopausal hormone receptorpositive women [8,9]. In addition, while epidemiological evidence has shown no association between the use of agents for dyslipidemia, mainly statins, and reduced breast cancer incidence, it supports a protective effect of these drugs on reducing breast cancer recurrence or mortality [10]. Moreover, several basic studies have shown that statins can suppress cancer cell proliferation, exert anti-angiogenic effects, and reduce the invasiveness and metastatic potential of breast cancer cells [11][12][13][14][15][16]. In addition, Crocetto et al. reported that the lipid alterations may be a potential tumor biomarker to detect bladder cancer, endocrine-related cancer, in clinical practice [17]; however, the relationship between lipid metabolism and immune activity in breast cancer patients has not been sufficiently investigated.
On the other hand, chemotherapy enhances antitumor immune responses [18,19]. Some studies have revealed that changes in the lymphocytic subpopulations after NAC can be used as prognostic markers in patients with breast cancer patients [20][21][22][23]. In contrast, other studies have examined the metabolic changes before and after chemotherapy in breast cancer patients and showed significant changes in lipid levels [24,25]. Furthermore, Tian et al. reported that NAC exerts an adverse effect on lipid levels during chemotherapy [26].
The present study investigated the correlation between lipid metabolism, antitumor immune responses, and chemosensitivity in patients with breast cancer treated with NAC.

Patient background
Data from the Osaka City University Graduate School of Medicine (Osaka, Japan) between April 2007 and March 2018 were analyzed. A total of 351 patients were diagnosed with early stage breast cancer (stage IIA, IIB, IIIA) and underwent with primary systemic treatment and curative surgery. We excluded 24 patients treated with neoadjuvant endocrine therapy and included 327 patients treated with NAC in this study. T and N factors and tumor stage were stratified based on the TNM Classification, UICC Seventh Edition [27]. Tumors were classified into intrinsic subtypes according to the immunohistochemical expression of the estrogen receptor (ER), progesterone receptor (PgR), and HER2. We defined ER + and/or PgR + and HER2-breast cancer as luminal, ER + and/or PgR + and HER2 + breast cancer as luminal-HER2, ER-and PgR-, HER2 + breast cancer as HER2-enriched, and ER-, PgR-, and HER2-breast cancer as TNBC. The antitumor effect was assessed according to the Response Evaluation Criteria in Solid Tumors [28]. The objective response (OR) was calculated as the sum of the clinical partial response and complete response (CR). All the patients underwent mastectomy or breastconserving surgery after NAC. The pCR was defined as the complete disappearance of the invasive compartment of the lesion with or without intraductal components, including the lymph nodes" [29]. Postoperative adjuvant therapy suitable for each intrinsic breast cancer subtype was performed, and standard postoperative radiotherapy to the remnant breast was administered if necessary. All patients underwent physical examinations, blood tests, ultrasonography, computed tomography, and bone scintigraphy scans. Overall survival (OS) was defined as the time from curative surgery to death from any cause, and recurrence-free survival (RFS) was defined as freedom from all locoregional and distant recurrences. The median follow-up time for the assessment of OS was 5.5 years (range 0.2-12.4 years) and for RFS was 4.9 years (range 0.1-12.0 years).

Blood sample analysis
Peripheral blood samples were obtained at the time of diagnosis, and preoperative blood samples were obtained within a week before surgery. We evaluated the serum lipid levels, including total cholesterol (TC) levels [categorized as low (≤ 149 mg/dl), normal (150-219 mg/ dl), and high (≥ 220 mg/dl)] and triglyceride (TG) levels [categorized as low (≤ 49 mg/dl), normal (50-149 mg/dl), and high (≥ 150 mg/dl)]. The differential white blood cell counts were analyzed using a Coulter LH 750 Hematology Analyzer (Beckman Coulter, Brea, CA, USA). The neutrophil-to-lymphocyte ratio (NLR) was calculated from the blood samples by dividing the absolute neutrophil count by the absolute lymphocyte count (ALC).

Statistical analyses
Statistical analyses were performed using the JMP13 software (SAS Institute, Cary, NC, USA). Associations among the variables were analyzed using the χ 2 or Fisher's exact test, as appropriate. OS and RFS were estimated

Clinicopathological responses of all the breast cancer patients to NAC
The differences in clinicopathological features due to intrinsic breast cancer subtypes are presented in Table 1  In all breast cancer patients, RFS and OS were significantly longer in patients who achieved pCR than in those who did not (P < 0.001 and P = 0.006, log-rank, respectively; Additional file 1: Fig. S1a, Additional file 2: Fig.  S2a). Furthermore, these outcomes were also significantly better in patients who achieved OR than in those who did not (P < 0.001 and P = 0.001, log-rank, respectively; Additional file 3: Figs. S3a, Additional file 4: Fig. S4a). In addition, we investigated the prognostic factors for RFS and OS for each breast cancer subtype. Among patients with luminal cancer, no significant differences were observed in RFS (P = 0.882, P = 0.399, log-rank, respectively) and OS (P = 0.861, P = 0.202, log-rank, respectively) according to the clinicopathological responses, pCR, and OR (Additional file 1: Fig. S1b, Additional file 2: Fig. S2b, Additional file 3: Fig. S3b, Additional file 4: Fig. S4b). In contrast, among the patients with TNBC, RFS (P = 0.005, P < 0.001, log-rank, respectively) and OS (P = 0.003, P < 0.001, log-rank, respectively) were significantly longer in patients who achieved pCR or OR than in those who did not (Additional file 1:

Analysis of relationships between lipid metabolism and chemosensitivity and prognosis
The relationship between lipid metabolism and chemosensitivity was examined (Table 2). There were no significant correlations between lipid metabolism and the pCR in any breast cancer subtype. In contrast, only TNBC patients with OR had significantly higher TG levels after NAC than patients without OR (P = 0.049).
We also investigated the prognostic value of serum lipid levels before and after NAC for each intrinsic breast cancer subtype. In patients with HER2-enriched breast cancer, those with normal TC levels before NAC had a significantly better OS than those with high TC levels (P = 0.013, log-rank test) (Fig. 1d), and in patients with TNBC, the group with high TC levels after NAC had significantly better OS than those with normal TC levels (P = 0.014, log-rank test) (Fig. 2e). There was no association between recurrence and TC levels. Also, there was no relationship between the prognosis and triglyceride levels before and after NAC (Additional files 5-10: Figs. S5-S10).

Analysis of relationships between lipid metabolism and immune activity
The pre-NAC ALC ranged from 712. 8 Table S1). The relationship between lipid metabolism and systemic immune activity is shown in Table 3. Patients with a high ALC before NAC had significantly higher TG levels after NAC in all the breast cancers (P = 0.001). In addition, among the patients with HER2-enriched breast cancer, high TG levels after NAC were associated significantly with a high ALC before NAC (P = 0.021), and high TG levels before NAC were associated significantly with a high ALC after NAC (P = 0.046). Furthermore, among patients with TNBC, high TG levels after NAC were associated significantly with a high ALC (P = 0.008) and a low NLR (P = 0.025) before NAC, while high TG levels before NAC were associated significantly with a low NLR after NAC (P = 0.034).

Discussion
In the present study, NAC increased serum TG levels, particularly in patients with TNBC. Some previous studies showed that serum lipid levels increased significantly after chemotherapy and that the TG levels may be a sensitive biomarker for determining the effect of adjuvant chemotherapy [24,30]. Many anticancer drugs are metabolized in liver and may cause non-alcoholic fatty liver disease by variety of mechanisms [31]. However, this phenomenon has not yet been fully studied. To the best of our knowledge, our study was the first to analyze the predictive value of lipid metabolism for chemosensitivity of breast cancer patients treated with NAC and to stratify the intrinsic subtypes of breast cancer.
In this study, patients with reduced tumor size had significantly higher TG levels after NAC in only TNBC. Sharma et al. reported that some chemotherapy agents affect serum lipid levels by regulating the expression of genes involved in lipid metabolism in liver cells [32]. Therefore, it is considered that there is a correlation between the effects of NAC and lipid metabolism in TNBC.
The efficacy of NAC, especially in terms of the pCR, is currently acknowledged as an indicator of good outcomes in patients with TNBC and HER2-enriched breast cancer, which have high immune activity [3,33,34]. Hence, it is expected that there will be an association between lipid metabolism and tumor immune activity in TNBC. Recent studies have reported that the regulation of metabolism can affect the tumor immune microenvironment and enhance the antitumor immune response [5][6][7]. In our study, good systemic immune activity, a high ALC, or low NLR before NAC were associated significantly with high TG levels after NAC in patients with TNBC or HER2-enriched breast cancer.
However, no relationships were observed between the pre-NAC lipid levels and NAC efficacy. In addition, the serum lipid levels before NAC showed no significant relationships with the ALC or NLR. Hence, it was difficult to predict chemosensitivity or systemic immune activity based on serum lipid levels prior to NAC.
In the present study, no significant associations were observed between the systemic immune activity and the effect of NAC. However, in our previous study, we set the cutoff value of pre-NAC NLR to 3.0, in the same breast cancer patients, and the pCR rate was significantly higher in TNBC patients with a good immune status, low NLR group [4]. This result suggested that not only the effect of tumor reduction, but also the effect of increasing serum lipid levels is recognized in patients with good systemic immune activity.
Although the TG levels after NAC may be an indicator of chemosensitivity in TNBC, they are not useful predictive markers of recurrence. The reason for this may be that changes in the lipid profiles after NAC are temporary [26]. We presumed that a favorable prognosis may not be based on lipid levels at the time of diagnosis or after NAC, but is induced by the maintenance good lipid metabolism after surgery.
This study has some limitations. First, this was a single-center, retrospective study, then the sample size was relatively small. Second, in our study, serum TC levels were associated with better OS in patients with HER2enriched breast cancer or TNBC. However, we did not have detailed data on high-density lipoprotein cholesterol and low-density lipoprotein cholesterol levels. In addition, many factors influence serum lipid levels, including lifestyle and adherence to medication. Considering these limitations, further prospective multicenter studies are needed.

Conclusions
This is the first study to demonstrate the clinical relationships between lipid metabolism, chemosensitivity, and systemic immune activity in patients with breast cancer